Summary: Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 240 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing due to immigration of persons from endemic regions. The natural history of chronic hepatitis B (CHB) also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B. Knowledge of the rate of progression between individuals with HBeAg positive and negative CHB is unknown. An equally important and related issue is the clinical assessment of disease severity in patients with CHB. Unfortunately, there are no good laboratory markers of disease severity. Liver biopsy is the accepted gold standard for assessing disease severity and cirrhosis but is costly, invasive, and associated with complications, which often limits patient acceptability as well as being subject to sampling error ranging from 15% to 20%. Non-invasive methods to assess disease severity are highly desirable for physicians caring for patients with CHB. Despite the availability of safe and effective oral nucleoside analogues for treatment of CHB, therapy remains problematic due to the need for prolonged therapy and limited effectiveness and tolerability of the alternate treatment, interferon. Clearance of hepatitis B surface antigen is the desired surrogate endpoint of therapy but is rarely achieved with current therapy. Identifying the optimal regimen, defining when to treat, for how long and when to stop therapy to achieve this endpoint are major unresolved issues. In addition, defining the best parameters to monitor patients both on and off therapy are not clear. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. To study this problem, we have analyzed a large database of untreated and treated patients with CHB (n750), focusing on the outcome of infection after hepatitis B e antigen (HBeAg) seroconversion. We have analyzed factors that predict progression to HBeAg negative chronic hepatitis B as well as factors that predict HBsAg loss. These analyses identified that 10% or greater of patients do not fit one of the traditional phenotypes used to classify the phase of HBV infection. The natural history of these subjects is being evaluated. The database is also being used to develop a non-invasive model to predict fibrosis progression in patients with CHB. We also plan to evaluate the role of transient ultrasound elastography (Fibroscan) to assess fibrosis stage in persons with CHB. These results will be compared to liver biopsy, MRI elastography and plasma will be stored for future proteomic analysis. The goal is to develop a series of blood and imaging tests that will obviate the need for liver biopsy in most patients with CHB. The Liver Diseases Branch is participating in a large multicenter study, the Hepatitis B Research Network, to define the natural history of HBeAg positive and negative chronic hepatitis B. This study has enrolled 2,000 patients at 13 North American sites. Primary questions that will be spearheaded by our group will be to identify the predictors of spontaneous HBeAg loss in immunetolerant patients and to evaluate the natural history of patients with elevated HBV DNA levels but normal ALT who are also HBeAg negative-so called indeterminate phenotype. We reported that diabetes was more prevalent in HBV-infected North American adults than the general population and is associated with known metabolic risk factors and liver damage, as determined by ALT levels. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. Relapse is common if treatment is discontinued after one year in the absence of HBsAg loss. Consequently, nucleos(t)ides must often be administered long-term or indefinitely. However, long-term use is associated with increased risk of side effects and higher costs. Therefore, the focus of current studies is to develop strategies to induce HBsAg loss (functional cure) to permit discontinuation of therapy and improve outcome of the infection. We are taking multiple approaches to this problem. The first is to compare the combination of tenofovir and emtricitabine compared to monotherapy with tenofovir for patients with CHB. Tenofovir is a nucleotide analogue that is more effective than adefovir at suppressing HBV DNA and has an excellent resistance profile. This study has enrolled 32 subjects and is ongoing. The second approach is to discontinue therapy in patients receiving long-term nucleos(t)ide analogues. To address this question we have an ongoing prospective trial to withdraw patients from medication and observe for benefits (HBsAg loss) or adverse events (ALT flares, HBeAg seroreversion and fulminant hepatitis). To date 15 patients have been withdrawn from therapy. The third approach is to combine peginterferon alfa with tenofovir compared to tenofovir alone. This is being conducted under the auspices of the hepatitis B research network and is a multicenter trial that enrolled 200 subjects and is ongoing. The fourth approach is to add peginterferon to ongoing long-term nucleos(t)ide analogue therapy for a period of 6 month. This trial will explore the mechanism of action of interferon during therapy of chronic hepatitis B. This trial has enrolled 13 subjects to date and is ongoing. 3) Elucidate the viral pathogenesis of HBV infection and mechanisms of anti-viral resistance The course of CHB is variable and affected by host, viral and environmental factors. We are investigating the role of pre-core and basic core promoter mutations on the outcome of chronic HBV infection after HBeAg serconversion. We hypothesize that elevation in HBV DNA levels are due to an increase in variant virus over wild type virus. We have quantified the proportion of pre-core and basic core promoter mutations relative to wild type virus among inactive carriers (HBV DNA <2000 IU/ml and normal ALT), indeterminate chronic HBV infection (HBV DNA >2000-<20,000 IU/ml and any ALT value) and HBeAg negative chronic hepatitis B (HBV DNA >20,000 IU/ml and ALT >2XULN). The results showed that the HBV phenotype was strongly related to the proportion of pre-core variant and ALT flares were more common when the proportion of either the pre-core and basic core promoter variants increased in the serum. This work was accepted for presentation and the molecular biology HBV meeting and the American Association for the Study of Liver Diseases meeting. The level of hepatitis B virus among the infected population is broad and ranges 9 log10 IU/ml. The cause for this is unknown. We are performing whole genome sequencing from patients that span the range of viral loads and patients with atypical HBV phenotype to identify mutations that may affect viral replication. Novel mutations identified in the sequence analysis will be engineered into a laboratory replicative construct (wildtype adwR9), using site directed mutagenesis. Replication competence of these mutant constructs will then be assessed after transient transfection of HuH-7 cells. Comparison of replication levels (measured by HBV DNA and HBsAg quantitation) will be made with wildtype adwR9 to determine whether these mutations upregulate or downregulate HBV replication. We are exploring potential mechanisms for clearance of HBsAg-the viral protein associated with clearance of HBV-by measuring the kinetics of HBsAg loss using a quantitative HBsAg assay. Finally we have performed an unbiased microRNA analysis and correlated this with HBV phenotype. Analysis is ongoing.